HARE-Mediated Endocytosis of Hyaluronan and Heparin Is Targeted by Different Subsets of Three Endocytic Motifs.

Pandey MS, Harris EN, Weigel PH - Int J Cell Biol (2015)

Bottom Line:
We previously found (Pandey et al.Biol.Single-motif deletion variants lacking M1, M3, or M4 (a different subset than involved in HA uptake) showed decreased Hep endocytosis, although M3 was the most active; the remaining redundant motifs did not compensate for loss of other motifs.

ABSTRACTThe hyaluronan (HA) receptor for endocytosis (HARE) is a multifunctional recycling clearance receptor for 14 different ligands, including HA and heparin (Hep), which bind to discrete nonoverlapping sites. Four different functional endocytic motifs (M) in the cytoplasmic domain (CD) target coated pit mediated uptake: (YSYFRI(2485) (M1), FQHF(2495) (M2), NPLY(2519) (M3), and DPF(2534) (M4)). We previously found (Pandey et al. J. Biol. Chem. 283, 21453, 2008) that M1, M2, and M3 mediate endocytosis of HA. Here we assessed the ability of HARE variants with a single-motif deletion or containing only a single motif to endocytose HA or Hep. Single-motif deletion variants lacking M1, M3, or M4 (a different subset than involved in HA uptake) showed decreased Hep endocytosis, although M3 was the most active; the remaining redundant motifs did not compensate for loss of other motifs. Surprisingly, a HARE CD variant with only M3 internalized both HA and Hep, whereas variants with either M2 or M4 alone did not endocytose either ligand. Internalization of HA and Hep by HARE CD mutants was dynamin-dependent and was inhibited by hyperosmolarity, confirming clathrin-mediated endocytosis. The results indicate a complicated relationship among multiple CD motifs that target coated pit uptake and a more fundamental role for motif M3.

fig4: HARE mediates the endocytosis of HA or Hep in the presence of M3 alone, but not in the presence of M2 or M4 alone. Cells expressing WT HARE (●), EV (○), or single-motif containing HARE CD variants +M2 (▼), +M3 (Δ in (a); ▽ in (b)), or M4 (□) were grown and treated as in Figure 2, incubated at 37°C for the indicated times with 125I-labeled Hep (a) or HA (b), and processed to quantify uptake as described in Methods section. Values are means ± SE (n = 9) and all linear regression lines had correlation coefficients ≥0.97. The three symbols for EV, +M2, and +M4 cells overlap at essentially identical positions in the bottom lines of each panel.

Mentions:
To address how multiple motifs function together to facilitate Hep endocytosis, we examined 125I-SA•ligand uptake in cells expressing different triple-motif deletions so that only single motifs remained (Figure 4). Interestingly, 125I-SA•b-Hep endocytosis by +M2 or +M4 cells was severely impaired by ≥95%. In contrast +M3 cells retained 65% of the HARE-specific endocytic capability of WT cells, an effect similar to the single-motif deletions ΔM1 or ΔM4. Based on studies with the single-motif deletion variants, especially ΔM3 cells, we expected that all three HARE CD variants containing only M2, M3, or M4 would be able to target HARE-Hep complexes to coated pits and mediate effective uptake. Since M2 does not participate in Hep uptake (Figure 3(a)), we expected +M2 cells to be defective in Hep endocytosis. However, the inability of +M4 cells to take up Hep was unexpected, since this motif is functional in WT cells. The unexpected functional differences among the single-motif containing HARE variants are not ligand specific, as the same pattern was observed when HA endocytosis was examined (Figure 4(b)). Again, +M2 cells (expected to be active; Figure 4(a)) or +M4 cells (expected to be inactive) were identical to EV cells; they were both unable to internalize HA, indicating the lack of coated pit targeting and uptake. In contrast +M3 cells showed ~60% of the endocytic capability of WT cells, a very similar result to that for Hep uptake (Figure 4(a)). Thus, although both HA (data not shown) and Hep (Figures 2(c) and 2(d)) bind equally well to HARE variants with only a single M2, M3 or M4 motif and these variants show similar surface-internal distributions (Figure 2), only M3 by itself is able to target HARE-ligand complexes to coated pits and mediate efficient uptake. The quantitative and relative rates of 125I-SA•b-Hep endocytosis of the various HARE CD mutants are summarized and compared to the values for HA uptake [26] in Table 1.

fig4: HARE mediates the endocytosis of HA or Hep in the presence of M3 alone, but not in the presence of M2 or M4 alone. Cells expressing WT HARE (●), EV (○), or single-motif containing HARE CD variants +M2 (▼), +M3 (Δ in (a); ▽ in (b)), or M4 (□) were grown and treated as in Figure 2, incubated at 37°C for the indicated times with 125I-labeled Hep (a) or HA (b), and processed to quantify uptake as described in Methods section. Values are means ± SE (n = 9) and all linear regression lines had correlation coefficients ≥0.97. The three symbols for EV, +M2, and +M4 cells overlap at essentially identical positions in the bottom lines of each panel.

Mentions:
To address how multiple motifs function together to facilitate Hep endocytosis, we examined 125I-SA•ligand uptake in cells expressing different triple-motif deletions so that only single motifs remained (Figure 4). Interestingly, 125I-SA•b-Hep endocytosis by +M2 or +M4 cells was severely impaired by ≥95%. In contrast +M3 cells retained 65% of the HARE-specific endocytic capability of WT cells, an effect similar to the single-motif deletions ΔM1 or ΔM4. Based on studies with the single-motif deletion variants, especially ΔM3 cells, we expected that all three HARE CD variants containing only M2, M3, or M4 would be able to target HARE-Hep complexes to coated pits and mediate effective uptake. Since M2 does not participate in Hep uptake (Figure 3(a)), we expected +M2 cells to be defective in Hep endocytosis. However, the inability of +M4 cells to take up Hep was unexpected, since this motif is functional in WT cells. The unexpected functional differences among the single-motif containing HARE variants are not ligand specific, as the same pattern was observed when HA endocytosis was examined (Figure 4(b)). Again, +M2 cells (expected to be active; Figure 4(a)) or +M4 cells (expected to be inactive) were identical to EV cells; they were both unable to internalize HA, indicating the lack of coated pit targeting and uptake. In contrast +M3 cells showed ~60% of the endocytic capability of WT cells, a very similar result to that for Hep uptake (Figure 4(a)). Thus, although both HA (data not shown) and Hep (Figures 2(c) and 2(d)) bind equally well to HARE variants with only a single M2, M3 or M4 motif and these variants show similar surface-internal distributions (Figure 2), only M3 by itself is able to target HARE-ligand complexes to coated pits and mediate efficient uptake. The quantitative and relative rates of 125I-SA•b-Hep endocytosis of the various HARE CD mutants are summarized and compared to the values for HA uptake [26] in Table 1.

Bottom Line:
We previously found (Pandey et al.Biol.Single-motif deletion variants lacking M1, M3, or M4 (a different subset than involved in HA uptake) showed decreased Hep endocytosis, although M3 was the most active; the remaining redundant motifs did not compensate for loss of other motifs.

ABSTRACTThe hyaluronan (HA) receptor for endocytosis (HARE) is a multifunctional recycling clearance receptor for 14 different ligands, including HA and heparin (Hep), which bind to discrete nonoverlapping sites. Four different functional endocytic motifs (M) in the cytoplasmic domain (CD) target coated pit mediated uptake: (YSYFRI(2485) (M1), FQHF(2495) (M2), NPLY(2519) (M3), and DPF(2534) (M4)). We previously found (Pandey et al. J. Biol. Chem. 283, 21453, 2008) that M1, M2, and M3 mediate endocytosis of HA. Here we assessed the ability of HARE variants with a single-motif deletion or containing only a single motif to endocytose HA or Hep. Single-motif deletion variants lacking M1, M3, or M4 (a different subset than involved in HA uptake) showed decreased Hep endocytosis, although M3 was the most active; the remaining redundant motifs did not compensate for loss of other motifs. Surprisingly, a HARE CD variant with only M3 internalized both HA and Hep, whereas variants with either M2 or M4 alone did not endocytose either ligand. Internalization of HA and Hep by HARE CD mutants was dynamin-dependent and was inhibited by hyperosmolarity, confirming clathrin-mediated endocytosis. The results indicate a complicated relationship among multiple CD motifs that target coated pit uptake and a more fundamental role for motif M3.